Planar optical waveguide with core of low-index-of-refraction interrogation medium

ABSTRACT

An apparatus for illuminating a sample includes a planar waveguide. The planar waveguide includes a first substrate, including a first outer surface and a first inner surface, and a second substrate, including a second outer surface and a second inner surface. The first and second inner surfaces of the first and second substrates, respectively, are spaced apart from each other and partly define a volume for confining the sample therein. The apparatus also includes a light source for providing light directed toward the planar waveguide, such that the light is optically coupled to and contained within the planar waveguide between the outer surfaces of the first and second substrates, while illuminating at least a portion of the sample confined within the volume.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a Continuation of U.S. patent applicationSer. No. 12/883,724, filed on Sep. 16, 2010, entitled PLANAR OPTICALWAVEGUIDE WITH CORE OF LOW-INDEX-OF-REFRACTION INTERROGATION MEDIUM,which is a Continuation-in-Part of U.S. patent application Ser. No.12/617,535, filed on Nov. 12, 2009, entitled WAVEGUIDE WITH INTEGRATEDLENS, which claims priority to U.S. Provisional Patent Application Ser.No. 61/156,586, filed on Mar. 2, 2009, entitled WAVEGUIDE WITHINTEGRATED LENS, all of which are incorporated by reference in theirentireties into the present application.

GOVERNMENT INTEREST

This research was funded in part by government support under the U.S.Department of Commerce National Institute of Standards (NIST) AdvancedTechnology Program (ATP), award number 70NANB7H7053. The Government hascertain rights in this invention.

BACKGROUND

Fluorescently labeled probes provide a convenient method ofcharacterizing the content of biological samples. By tailoring thebinding chemistry of a fluorescent probe, high specificity can beachieved for detection of complex molecules such as RNA, DNA, proteins,and cellular structures. Since fluorophores typically absorb and re-emitStokes-shifted radiation regardless of being bound or unbound to aspecies to be detected, the bound and unbound fluorophores must beseparated.

One common method to separate the bound fluorophores from the unboundfluorophores relies on spatial localization of the fluorescently labeledspecies. For example, in a ‘sandwich immunoassay,’ a surface ischemically treated to bind a species to be detected to that surface. Thefluorescent probes then attach to the species that are bound to thesurface. Unbound fluorophores can then be removed from the system with awash step.

Background fluorescence can be further reduced if the excitation lightcan be confined to the surface. Total internal reflection fluorescence(TIRF) is one method of reducing background fluorescence. In general,when light propagates from one medium to another, a portion of the lightwill be reflected at the interface. If the light is propagating into amaterial with a lower index of optical refraction, however, all of thelight will be reflected if the angle at which the beam is incident onthe surface is greater than the ‘critical angle’ (relative to thesurface normal). In the lower index material, the light intensityexponentially decays with distance from the surface. This exponentiallydecaying field (known as an ‘evanescent field’) has a characteristicdecay length on the order of 100 nanometers to 1 micrometer for visiblelight. The light of the evanescent field will, therefore, only excitefluorophores that are localized at the surface.

In a simplified implementation, TIRF is performed with a laser beamreflecting once from the surface. This is the basis of well establishedTIRF microscopy and other biosensing techniques. By confining the laserbeam inside a waveguide, however, multiple reflections can be realizedand larger areas can be illuminated. Several waveguide geometries arepossible, each having certain tradeoffs.

Single-mode planar waveguides, also called thin film waveguides orintegrated optical waveguides, confine light into a small crosssectional area with the thin dimension smaller than the wavelength ofpropagating light. The advantage of single-mode waveguides is thatsignificantly stronger evanescent fields are generated. A disadvantageof single-mode waveguides is that for efficient light coupling, theytypically require a prism or grating with precise alignment tolerances.In addition, single-mode planar waveguides are expensive to manufacturebecause the guiding layer is typically a thin-film with strict thicknesstolerances deposited on a substrate. In contrast, a multimode planarwaveguide is substantially easier to couple a laser beam to and simplerto construct than single-mode planar waveguides. For example, a standard1 millimeter thick microscope slide makes an effective waveguide intowhich light can be coupled through the edge of the slide. Additionally,dimensions for multimode waveguides are compatible with current plasticinjection-molding techniques.

For a fluorescence-based assay system, a uniform evanescent field isdesired in the detection region. By definition, the strength of theevanescent field is uniform along the direction of light propagation fora single-mode planar waveguide (neglecting scattering losses andabsorption inside the waveguide). For a disposable clinical device,however, cost, robustness, and ease of use are of similar importance. Byadjusting input coupling to a multimode waveguide, uniformity and fieldstrength of the evanescent field can be optimized.

While each individual mode in a multimode waveguide has a uniformintensity along the direction of propagation, a distribution of modeswill be excited when coupling to a multimode waveguide; thisdistribution of modes will constructively and destructively interfere onthe surface and lead to a spatially varying field strength. When thethickness of the waveguide is much larger than the wavelength of light,the mode structure of the waveguide can be neglected, and the intensityin the waveguide can be treated as a conventional diffracting beam thattotally-internally reflects from the two surfaces of the waveguide andinterferes with neighboring reflections.

FIG. 1 illustrates several examples of existing coupling schemes 105-115involving multimode waveguides. Coupling scheme 105 using a multimodewaveguide 120 involves focusing a laser beam 125 that propagatesparallel to a waveguide 120 into the edge of waveguide 120 with acylindrical lens 130. The field strength of a total internal reflection(“TIR”) beam, however, is maximized for a beam that is incident at thecritical angle and zero for a beam with an incident angle 90° from thesurface normal (i.e., grazing incidence). Thus, an incident beam that isparallel to the TIR surface will have small evanescent field strengthwhen coupled to waveguide 120 with cylindrical lens 130 in theconfiguration of the scheme 105.

A variation on coupling scheme 105 is illustrated by coupling scheme110. In coupling scheme 110, a laser beam 135 focused by a cylindricallens 140 is incident on the edge of a waveguide 145 with an appropriateangle such that a central ray of laser beam 135 inside the waveguideimpinges on the surface near the critical angle for TIR to maximize theevanescent field strength. A compromise between field strength anduniformity may be made by the choice of focusing optics. If a nearlycollimated beam is used to achieve high field intensity by operatingnear the critical angle for TIR, the beam must make many reflectionswithin the waveguide before the surface intensity becomes sufficientlyuniform, thus requiring a longer waveguide. If the beam is highlyfocused, however, then the surface intensity normalizes in very fewreflections, but a significant amount of power is contained in rayspropagating outside the critical angle and leads to reduced evanescentfield strength down the length of the waveguide.

Precise alignment of a cylindrical lens, such as lenses 130 and 140,relative to the input face of a waveguide, such as waveguides 120 and145, respectively, must be made in order to have a laser beam focused onthe input face. One proposed solution to this problem is illustrated bya coupling scheme 115. In coupling scheme 115, a lens 150 isincorporated with a waveguide 155 as a single optical component, made,for example, by bonding the lens element to the planar waveguide or bymolding a single optical component. While this allows the focus of lens150 to be precisely distanced from the edge of waveguide 155, carefulalignment of a laser beam 160 relative to lens 150 of waveguide 155 muststill be made to couple beam 160 to waveguide 155. For applicationsrequiring repeated placement of a waveguide component relative to thelight source, it is highly desirable for the light coupling to berelatively insensitive to misalignment.

In practical applications, the penetration depth of the evanescent fieldusually is less than a wavelength of the incident light. This aspect isan advantage in some applications, as the evanescent field can serve asa mechanism to illuminate only a volume of interest, e.g., a thin layerin the lower refractive index medium proximate to the waveguide surface.On the other hand, when the object of interest, such as a cell or thebulk of a solution, extends substantially beyond the penetration depthof the evanescent wave, evanescent illumination can be less effectivethan floodlight-type illumination.

A subfield of integrated optofluidics is concerned with the developmentof methods for using optical waveguides to illuminate extended liquidmedia. Most of the developed methods involve the containment of a liquidsample by other liquid and/or solid materials, thereby effectivelycreating a waveguide for illuminating the liquid sample. Most TIR-baseddesigns involve surrounding the liquid sample with media of lower indexof refraction than that of the liquid sample itself. It is thentheoretically possible for light to be guided in the liquid sample byTIR at the interface between the high refractive index liquid and thelower refractive index surroundings. However, in practice, waveguidingin a liquid sample contained in another material is difficult due to thefact that common liquids have lower refractive indices than commonsolids; for example, water has a refractive index of approximately 1.33,while most solid materials have an index of refraction of 1.4 or more.Consequently, a majority of the TIR waveguide designs involve usingeither high refractive index (i.e., “high-n”) liquids or more exotic lowrefractive index (i.e., “low-n”) solids.

In interference-based optofluidic waveguides, light is confined to aliquid core by reflection from surrounding materials including two ormore layers of higher-index materials combined to result in a lowereffective refractive index for the surrounding media. Someinterference-based optofluidic waveguides include photonic crystals,such as multiple alternating layers of materials of different indices ofrefraction

SUMMARY OF THE CLAIMED INVENTION

Embodiments disclosed below allow light to be coupled to a planarwaveguide providing a strong evanescent field for sample illumination,while eliminating or greatly reducing inadvertent misalignment by auser. The various embodiments further allow facile tuning of theinternal propagation angle inside the waveguide, providing simpleadjustment of evanescent field strength. Another embodiment alsoprovides apparatus for performing assays involving placement of afluidic chamber on a planar waveguide in a manner that is insensitive tothe optical properties of the chamber.

In an embodiment, apparatus for illuminating a sample for analysis isdisclosed. The apparatus includes a light source, a planar waveguide,and a refractive volume. The light source provides light along apropagation vector. The planar waveguide is oriented such that thepropagation vector is perpendicular to the normal vector of the planarwaveguide and offset from the planar waveguide in a direction parallelto the normal vector of the planar waveguide. The refractive volume,which is positioned proximate to the planar waveguide, optically coupleslight provided by the light source to the planar waveguide.

Another embodiment sets forth a method for performing sample analysis.Light is provided from a light source along a propagation vector. Arefractive volume positioned proximate to a planar waveguide isilluminated with the light. The waveguide is oriented such that thepropagation vector is perpendicular to the normal vector of the planarwaveguide and offset from the planar waveguide in a direction parallelto the normal vector of the planar waveguide. The light is then coupledto the planar waveguide via the refractive volume.

Apparatus for performing biological assays is disclosed in yet anotherembodiment. The apparatus includes a light source, a planar waveguide, arefractive volume, and a detector. The light source provides light alonga propagation vector. The planar waveguide has a plurality of specificbinding molecules bound to a face thereof. The planar waveguide couldfurther have an array of two or more dissimilar specific bindingmolecules bound to the face thereof. Additionally, the optical axis ofthe planar waveguide is oriented parallel to the propagation vector andoffset from the propagation vector in a direction perpendicular to aface of the planar waveguide. The refractive volume optically coupleslight provided by the light source to the planar waveguide and ispositioned proximate to the planar waveguide. The refractive volumeincludes at least a section of a plano-convex cylindrical lens. Thedetector is positioned to detect light emitted from a region proximateto the face of the planar waveguide having the plurality of specificbinding molecules bound thereto.

In an embodiment, an apparatus for illuminating a sample includes aplanar waveguide. The planar waveguide includes a first substrate, witha first outer surface and a first inner surface, and a second substrate,with a second outer surface and a second inner surface. The first andsecond inner surfaces of the first and second substrates, respectively,are spaced apart from each other and partly define a volume forconfining the sample therein. The apparatus further includes a lightsource for providing light directed toward the planar waveguide suchthat the light is optically coupled to and contained within the planarwaveguide between the outer surfaces of the first and second substrates,while illuminating at least a portion of the sample contained within thevolume.

In a further embodiment, the sample contains at least one object, andthe planar waveguide and the light source are configured to cooperate touniformly illuminate the object. In a still further embodiment, theobject is greater than one micrometer in diameter.

In a yet further embodiment, the apparatus further includes a gasket forseparating the first and second inner surfaces of the first and secondsubstrates, respectively, while further defining the volume forconfining the sample therein. In a further embodiment, the light iscontained between the outer surfaces of the first and second substratesat least in part by total internal reflection. In a still furtherembodiment, the light source provides uncollimated light.

In another embodiment, a sample analysis system includes a planarwaveguide. The planar waveguide in turn includes a first substrate, witha first outer surface and a first inner surface, and a second substrate,with a second outer surface and a second inner surface. The first andsecond inner surfaces of the first and second substrates, respectively,are spaced apart from each other and partly define a volume forconfining a sample therein. The sample analysis system further includesa first light source for providing a first illumination directed towardthe planar waveguide. The first illumination is optically coupled to andcontained within the planar waveguide between the outer surfaces of thefirst and second substrates while illuminating at least a portion of thesample confined within the volume. The sample analysis system alsoincludes a detector for detecting a first light signal emitted from thesample as a result of the first illumination interacting with theportion of the sample.

In a further embodiment, the sample analysis system includes a secondlight source, which is configured for providing a second illumination,and imaging optics for directing the second illumination from the secondlight source to at least another portion of the sample and to thedetector. The detector is further configured for detecting a secondlight signal resulting from the second illumination interacting with theat least another portion of the sample.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates several examples of existing coupling schemesinvolving multimode waveguides.

FIG. 2 illustrates a generalized configuration descriptive of exemplaryembodiments.

FIG. 3 illustrates a cross-sectional view of an exemplary waveguide withan integrated lens.

FIG. 4 provides a detailed cross-sectional view of the waveguide withthe integrated lens depicted in FIG. 3.

FIG. 5 is a cavalier projection view illustrating the exemplarywaveguide with the integrated lens.

FIG. 6 is a cavalier projection view illustrating an exemplary gasketwith multiple channels.

FIG. 7 is a flowchart of an exemplary method for performing sampleanalysis.

FIGS. 8 and 9 illustrate an exemplary embodiment of a planar low-n corewaveguide, with liquid sample containment by two-dimensional surfacetension. Within the context of the present disclosure, a planar low-ncore waveguide is a planar waveguide in which the core of the waveguideexhibits a lower refractive index than the materials that surround thecore.

FIGS. 10 and 11 illustrate an exemplary embodiment of another planarlow-n core waveguide, with sample containment by a solid sealingmaterial for four sides and surface tension for two sides.

FIGS. 12-15 are illustrations of light propagating in an embodiment of aplanar low-n core waveguide. FIGS. 12 and 13 show collimated lightpropagating through thick and thin waveguides. FIGS. 14 and 15 showdiverging light in thick and thin waveguides. In all figures, partialreflections at various internal interfaces (e.g., thesubstrate-to-interrogation medium interface) have been omitted forclarity.

FIGS. 16-25 show diagrammatic illustrations of variations for lightcoupling means suitable for use with the planar low-n core waveguide.

FIG. 26 shows an exemplary embodiment, in which the interrogation mediumis fully contained by solid material, and the light is coupled into thewaveguide itself within the containment region. The dashed linerepresents an exemplary shape for appropriate light coupling into thewaveguide.

FIGS. 27-28 illustrate exemplary embodiments of a planar low-n corewaveguide with a liquid interrogation medium, including light couplingmeans, fluid containment, and fluid inlet and outlet ports. Bothsubstrates may be optically clear for the wavelength range of interest,as shown in FIG. 28. A gasket may be used to contain the liquid in twodimensions, as shown in FIGS. 27-28. Alternatively, the upper componentmay be shaped to include side walls or stand-offs, which may be directlybonded to the waveguide substrate.

FIGS. 29 and 30 show embodiments similar to that shown in FIG. 28,wherein the upper substrate further includes a reflector.

It is noted that, for purposes of illustrative clarity, certain elementsin the drawings may not be drawn to scale.

DETAILED DESCRIPTION

Embodiments of the present technology provide for sample illuminationsuch as that involved in fluorescence detection and assay based onevanescent fields using apparatus including a waveguide with anintegrated lens. The overall configuration of the apparatus may be suchthat fluorescence-emitting molecules bound to a waveguide surface areexcited by an evanescent field penetrating into the adjacent solutionfrom a light beam propagated within the waveguide, the propagated beambeing introduced by an integrally connected lens. The collimated beam oflight such as a laser beam may propagate parallel to the waveguidesurface such that the system is insensitive to translation of thewaveguide. The incident beam may be also appropriately offset from theoptical axis of the waveguide such that refraction of the light at thelens surface directs the beam into the waveguide at an angle close tothe critical angle for TIR. Additionally, a second integratedcylindrical lens may be added to the output end of the waveguide. Thisaddition of the second integrated cylindrical lens may facilitate asecond laser being coupled in the opposite direction, such as for use inmulti-color fluorescence assays.

The apparatus may also allow a fluidic chamber to be bound to the planarwaveguide such that the chamber contact with the planar waveguide isoutside the optical path of the propagating light, eliminatingrestrictions on optical properties of material comprising the chamber.In some previous configurations, fluidic chambers have utilized lowindex of refraction materials in contact with the planar waveguide withmechanical clamping in order to limit optical losses at thewaveguide/chamber contact area. By separating the waveguide/chambercontact from the optical path, traditional bonding methods such asadhesives or plastic welding may be used to attach the chamber to thewaveguide. Moreover, the fluidic chamber may include or be formed inpart by a second planar waveguide, wherein the fluidic chamber isdisposed between two planar waveguides. In such an arrangement light maybe coupled to both planar waveguides as well as the volume formed by thefluidic chamber.

FIG. 2 illustrates a generalized configuration 200 descriptive ofexemplary embodiments. Configuration 200 includes a light source 205, arefractive volume 210, and a planar waveguide 215. Light source 205 caninclude a laser or any other source of collimated or near-collimatedlight that provides light along a propagation vector 220. Refractivevolume 210 is positioned proximate to planar waveguide 215. Refractivevolume 210 and planar waveguide 215 may lack a discontinuity in index ofrefraction therebetween. For example, refractive volume 210 may beadjacent to or abutted to waveguide 215 with an index matching fluid(not shown) occupying any gap therebetween. Alternatively, refractivevolume 110 may be integrated with planar waveguide 215 as a single unitor article. Planar waveguide 215 is oriented such that propagationvector 220 is perpendicular to normal vector 225 of planar waveguide215. Furthermore, planar waveguide 215 has an offset 230 in a directionparallel to the normal vector 225 of planar waveguide 215.

FIG. 3 illustrates an exemplary cross-sectional view 300 of a waveguide305 with an integrated lens 310 according to one embodiment.Additionally, view 300 depicts a collimated light beam 315 such as thatof a laser with a wavelength appropriate to excite fluorescent probes atan assay surface 320. Planar waveguide 305 with integrated lens 310 isconfigured to inject collimated light beam 315 through a bottom surfaceof planar waveguide 305. A flowcell is formed from a sealing mechanism,such as a gasket 325, an inlet port 330, an output port 335, and afluidic sample chamber 340, in which chemical compounds deposited onassay surface 320 of waveguide 305 may bind the desired target compoundto the surface. Collection and filtering optics 345 can capturefluorescence from assay surface 320 of waveguide 305. A signalcorresponding to the fluorescence so captured may then be directed to animaging device 350 such as a CCD or CMOS camera. Furthermore, the roof,the floor, and/or the walls of the flow cell may be used as a surface onwhich compounds are deposited.

It is noteworthy that fluidic sample chamber 340 may include or beformed in part by a second planar waveguide, similar to waveguide 305,such that fluidic sample chamber 340 is disposed between two planarwaveguides. In such a configuration, light may be coupled to bothwaveguide 305 and the second planar waveguide as well as the volumeformed by the fluidic sample chamber 340. The principles describedherein are similarly applicable to configurations having multiple planarwaveguides.

FIG. 4 provides a detailed cross-sectional view 400 of waveguide 305with integrated lens 310. For further reference, FIG. 5 is a cavalierprojection view 500 illustrating waveguide 305 with integrated lens 310.Referring back to FIG. 4, collimated light beam 315 propagates in adirection parallel or nearly parallel to the optical axis of waveguide305, but offset from the optical axis such that it strikes the curvedsurface of integrated lens 310. For a clinical instrument in which thewaveguide structure is a removable consumable item, this geometry mayloosen the positional tolerances necessary to couple collimated lightbeam 315 reproducibly to waveguide 305. Collimated light beam 315impinges on the curved surface of integrated lens 310 at a non-zeroangle α relative to the local surface normal of integrated lens 310, asillustrated in FIG. 4.

As a result of refraction explained by Snell's law, collimated lightbeam 315 refracts such that it strikes the top surface of waveguide 305at an angle β relative to the optical axis of waveguide 305. The angle βis defined as the internal propagation angle. The vertical distance ybetween the center of collimated light beam 315 and the apex ofintegrated lens 310 is chosen such that β is less than the complement ofthe critical angle allowing total internal reflection to occur. For agiven radius R for the curved surface of integrated lens 310 and indexof refraction n for integrated lens 310, the distance y and angle β arerelated by the equation:

$\begin{matrix}{y = {{R\left\lbrack {1 - \frac{n\; \sin \; \beta}{\sqrt{1 - {2n\; \cos \; \beta} + n^{2}}}} \right\rbrack}.}} & \left\lbrack {{Eq}.\mspace{14mu} 1} \right\rbrack\end{matrix}$

Since collimated light beam 315 has a spatial extent, the curved surfaceof integrated lens 310 will act to focus collimated light beam 315. Theradius R of the curved surface of integrated lens 310 is chosen suchthat for a given beam diameter of collimated light beam 315, the rangeof angles incident on the top surface of waveguide 305 is appropriate toprovide a uniform evanescent field strength within the detection regionwhile remaining outside the critical angle for TIR. It may be desiredthat collimated light beam 315 be focused on the top surface thewaveguide 305 to allow for the greatest tolerance to misalignment. Thetotal thickness t for the structure formed from waveguide 305 andintegrated lens 310 that leads to a focused beam on the top surface maybe given by:

$\begin{matrix}{t = {R + {\frac{\left( {y - R} \right)^{3}}{R^{2}n^{2}}.}}} & \left\lbrack {{Eq}.\mspace{14mu} 2} \right\rbrack\end{matrix}$

When an appropriate thickness t is used, collimated light beam 315 willfocus at a horizontal distance L from the center of the circle definingthe curved surface of integrated lens 310. L may be related to thepreviously defined quantities by the equation:

$\begin{matrix}{L = {\frac{t - y}{\tan \; \beta} - {\sqrt{{2{yR}} - y^{2}}.}}} & \left\lbrack {{Eq}.\mspace{14mu} 3} \right\rbrack\end{matrix}$

The structure including waveguide 305 and integrated lens 310 may bemanufactured in several different ways. One method is to have the entireassembly constructed in plastic by injection molding technology. Analternative method is to fabricate the planar waveguide and lens elementseparately from similar index materials. The two elements may then bejoined permanently by a transparent optical cement, optical contacting,or temporarily with index matching fluid/oil/gel.

Geometries such as those described in connection with FIG. 3 easilyallow the adjustment of the internal propagation angle (β) through atranslation, rather than a rotation, of the incident laser beam. Thisallows for a less complicated mechanical design to couple the laser tothe waveguide. Additionally, a new injection molded waveguide is notnecessary when it is desired to change the incident angle because thefocal point of the lens using the disclosed geometry of FIGS. 3 and 4 isinsensitive to the translation of a laser beam relative to the opticalaxis of waveguide 305. Further, a desired change in the incident angleis accomplished without changing the readout instrument, allowingvariation of cartridge function without physical changes in theinstrument. A barcode on the cartridge may be utilized to identifyinformation used to interpret signals from a given cartridge.

To prevent light from leaking from the waveguide 305 after the firstreflection from the top surface, the cylindrical lens 310 is truncatedsuch that it does not extend beyond the location of the focus. The areadefined by the line connecting the apex of integrated lens 310 and thepoint on the bottom surface opposite the focus (see, e.g., ‘opticaldeadzone 355 in FIG. 3) will never have light propagate in it thatsuccessfully couples to the waveguide. As such, the precise shape of thelens in the area designated optical deadzone 355 can be any convenientshape provided integrated lens 310 does not extend beyond the verticalline passing through the focus. For a single injection molded devicewhere minimizing material costs is important, removing all plastic inthe area labeled optical deadzone 355 may be desirable. If two separatecomponents made through conventional optical manufacturing processes arefabricated, integrated lens 310 that has been diced to remove materialbeyond the focus can be easily manufactured. A material that has lowautofluorescence properties may be desirable to minimize backgroundcontributions in the signal collection.

Because integrated lens 310 is used in off-axis geometry, minor opticalaberrations at the focus may be exhibited if the curved surface iscircular. While a circular profile functionally works, the use of anaspheric surface may be employed to extend the range of the verticalposition of the incident beam for which the beam will be coupled towaveguide 305, allowing a larger range of adjustment of the angle β. Theappropriate deviation from a circular profile can be calculated withoptical ray tracing programs familiar to those skilled in the art.

The large area of the top surface of waveguide 305 before the focus mayallow for a sample chamber to be sealed. Gasket 325 sealing surface maybe absent from the optical path. Therefore, a larger range of gasketmaterials may be possible that only need to be evaluated for theirchemical/biological compatibility and not their optical properties. Forexample, an adhesive backed spacer can be utilized to form a sealedflowcell without a complicated clamping mechanism. Multiple flow cellscan also be incorporated into a single biosensor by utilizing a gasketwith multiple channels.

FIG. 6 is a cavalier projection view illustrating an exemplary gasket605 with multiple channels. The width of each channel may be chosen tomatch the unfocused dimension of the incident beam such that lightcoupling to the gasket along the length of the waveguide is minimized. Amechanism for translating the incident beam between channels may beincluded. In addition, the top surface of waveguide 305 within the flowchannels may be appropriately treated to allow for the capture offluorescently labeled target molecules such as proteins, RNA, DNA, orcellular structures.

A lid attached to the gasket completes the flow cell. Fluid samples canbe introduced through orifices in the lid and flow through the channels,allowing the fluid to interact with the top waveguide surface. Fluidreservoirs exterior to the flow channel can also be included to allowthe introduction of fluids into the flow channel and an overflowreservoir at the outlet port of the flow channel to contain the fluidafter it has passed through the flow channel. With plastic components,the gasket may be optionally eliminated by molding the channels into oneof the plastic components and joining the two plastic componentsdirectly with methods known to those skilled in the art (e.g., laser orultrasonic welding).

The evanescent field created by the light within waveguide 305 canexcite fluorophores that have attached to the top surface of waveguide305. As the fluorophores relax and emit frequency shifted radiation, theemitted light may be captured by a lens or series of lenses (e.g.,collection and filtering optics 345) to transfer an image of the surfaceto a plane that is imaged by a light capturing device (e.g., imagingdevice 350) such as a CCD or CMOS sensor. An optical filter may also beplaced between the waveguide surface and the imaging device to eliminatescattered incident light that has not been frequency shifted by thecaptured fluorophores.

FIG. 7 is a flowchart of an exemplary method 700 for performing sampleanalysis. The steps of exemplary method 700 may be performed in varyingorders. Furthermore, steps may be added or subtracted from exemplarymethod 700 and still fall within the scope of the present technology.The methodology illustrated in FIG. 7 may be performed for fluorescencedetection and assay based on evanescent fields.

In a step 705, light is provided from a light source along a propagationvector. The light source may include a laser or any other source ofcollimated or near-collimated light.

In a step 710, a refractive volume is illuminated with the light. Therefractive volume is positioned proximate to, and may be integratedwith, a planar waveguide. In exemplary embodiments, the refractivevolume may include at least a section of a plano-convex cylindricallens, wherein the longitudinal axis of the refractive volume is orientedperpendicular to the optical axis and the normal vector of the planarwaveguide.

In a step 715, the light is coupled to the planar waveguide via therefractive volume. The waveguide is oriented such that the propagationvector is perpendicular to the normal vector of the planar waveguide andoffset from the planar waveguide in a direction parallel to the normalvector of the planar waveguide.

In an optional step 720, indicated by a dashed box, the optical couplingof the light provided by the light source to the planar waveguide istuned by translating the light source in a direction parallel to thenormal vector of the planar waveguide.

In a step 725, consistent optical coupling of the light provided by thelight source to the planar waveguide is maintained while translating thelight source parallel to the optical axis of the planar waveguide.

In a step 730, a biological sample is positioned in a reservoir formedat least in part by a face of the planar waveguide.

In a step 735, light emitted from a region proximate to a face of theplanar waveguide is detected. In some embodiments, a detector ispositioned to detect light emitted from a region proximate to the faceof the planar waveguide having a plurality of capture molecules boundthereto.

For some applications, containment of the liquid layer within asub-wavelength extent, as in the context of the applications describedabove, may be unfeasible. For instance, if the object of interest is abiological cell on the order of one to twenty microns in diameter, thena different approach to analyte illumination and light guiding isrequired.

Another important aspect to consider when designing optical waveguidesfor a practical application is the manufacturability of the waveguide,especially if the application is intended to enter volume productionwith cost requirements. The sensitivity to manufacturing tolerances mustbe evaluated as it can greatly influence the manufacturability and, inthe worst case, render the design unfeasible. Likewise, the method forcoupling light into the waveguide should be considered, since thelight-insertion method may impact both the waveguide manufacturabilityand the engineering effort required to interface the waveguide with thelight source. This issue is of particular concern if the light sourcewill not be permanently affixed to the waveguide. Additionally, theinterfacing complexity tends to increase as the waveguide dimensionsdecrease.

Although the coupling of light into micrometer-scale waveguides has beenimplemented in, for instance, telecommunications equipment, theengineering effort and manufacturing expenses are important factors tobe considered for cost-sensitive applications outside oftelecommunications. For instance, the various types of waveguidesdescribed above are generally inappropriate for mass production due totheir complexity.

It would be desirable to use an optical waveguide to efficientlyilluminate low-n media and/or objects embedded in such media, where themedia or objects extend beyond the penetration depth of the evanescentfield generated at a high-n to low-n interface. A low-n medium may be,for example, a material having an index of refraction lower than that ofconventional solid materials, e.g., a refractive index less than^(˜)1.5. An optical waveguide capable of effectively illuminating a corecontaining a low-index of refraction medium is described herein. It isnoted that the terms “light” and “illumination” are used interchangeablyherein.

In an embodiment, as illustrated in FIG. 8, a planar waveguide 800includes a stack of layers formed from a first substrate 802 and asecond substrate 804 sandwiching a low-n medium 810. The low-n medium isinterchangeably denoted herein as the interrogation medium. First andsecond substrates 802 and 804 may be, for instance, optically clear soas to be transparent to light having a wavelength within a predeterminedrange. Low-n medium 810 is introduced between first and secondsubstrates 802 and 804 such that first and second substrates 802 and 804cooperate to confine low-n medium 810 therebetween. First and secondsubstrates 802 and 804 and low-n medium 810 may have a variety ofthicknesses, as long as low-n medium 810 exhibits a lower refractiveindex in comparison to first and second substrates 802 and 804. Thepresent concept is compatible with numerous schemes of coupling lightinto the waveguide, as well as different methods of containing the low-nmedium therein. The low-n medium may be liquid, gaseous and/or solid.

One-dimensional optical confinement (i.e., in a direction indicated by asurface normal 820, indicated by a thick arrow, of the first and secondsubstrates) of light inserted into the waveguide may be provided by TIRat the interfaces between the optically clear substrates and theexternal surroundings. In the exemplary embodiment shown in FIG. 8, alight source 830 directs illumination 835 into planar waveguide 800 atan angle away from the substrate normal and out of the plane of thesubstrates such that one-dimensional optical confinement of illumination835 is provided by planar waveguide 800 by total internal reflection atthe two substrate-to-surrounding medium interfaces.

A cross-sectional view of planar waveguide 800 is shown in FIG. 9. Itshould be noted that the figures are not drawn to scale. As shown inFIG. 9, first substrate 802 has a refractive index n_(s1), secondsubstrate 804 has a refractive index n_(s2), and low-n medium 810 has arefractive index n_(m). Planar waveguide 800 is surrounded by air (orsome other medium) with a refractive index n_(a). The indices ofrefraction fulfill the requirements:

n_(a)<n_(s1),n_(s2)  [Eq. 4] and

n_(a)<n_(m).  [Eq. 5]

Note that critical angle for (θ_(1,2))_(c) for light propagation from afirst material (with refractive index n₁) toward a second material (withrefractive index n₂, where n₂<n₁) is given by:

$\begin{matrix}{\left( \theta_{1,2} \right)_{c} = {\arcsin \left( \frac{n_{2}}{n_{1}} \right)}} & \left\lbrack {{Eq}.\mspace{14mu} 6} \right\rbrack\end{matrix}$

As shown in FIG. 9, light 835 enters planar waveguide 800 such that anincidence angle θ_(s-a) from first substrate 802 (with refractive indexn_(s1)) into the surrounding medium (with refractive index n_(a)) isgreater than the critical angle (θ_(s,a))_(c) as defined from the lowerof n_(s1) and n_(s2), i.e.,

θ_(s,a)>(θ_(s,a))_(c),  [Eq. 7]

such that light 835 is contained within planar waveguide 800 by TIR. Allangles are measured relative to surface normal 820. Consequently, thesubstrates and the interrogation medium form a multi-part waveguide,together providing light confinement in one dimension (i.e., in adirection parallel to surface normal 820). The interrogation medium canbe of any type (e.g., gaseous, liquid, and biological objects embeddedin a liquid) as long as the refractive index condition of Eq. 4 andincidence angle condition of Eq. 7 are satisfied.

For liquid and gaseous interrogation media, the waveguide design may bemodified for containing the interrogation medium. For example, in theembodiment shown in FIGS. 8 and 9, low-n medium is contained betweenfirst and second substrates 802 and 804 entirely by surface tension.FIGS. 10 and 11 show an alternative configuration for a planar waveguide900, in which first and second substrates 802 and 804 are spaced apartby first and second gaskets 906 and 908. Still alternatively, first andsecond gaskets 906 and 908 may be connected to form a single contiguousgasket. It is noted that the embodiments shown in FIGS. 8-11accommodates the addition of inlet and outlet ports (not shown) for thelow-n, interrogation medium. The open ends in FIGS. 10 and 11 may beplugged using another material, thereby forming a completely-sealedvolume for containing the interrogation medium.

The containment configuration should be compatible with the method forcoupling light into the waveguide. For instance, the system may beconfigured such that the interrogation medium may be uniformlyilluminated in the plane of the planar waveguide, even if the light isnot solely confined within the interrogation medium. In-coupling oflight 835 through the substrates is generally unaffected by the low-nmedium containment schemes shown in FIGS. 8-11. Interference effects orcurved interface effects (e.g., if light 835 is incident from thesurrounding medium directly onto low-n medium 810, which may include aninterface curvature caused by surface tension) may affect subsequentpropagation of light 835 through planar waveguide 800 or 900.

Referring to FIG. 9, the illumination strength inside low-n medium 810depends on the angle of light propagation inside planar waveguide 800.Due to the spatial compression of the light reflection at the, lightpropagating at angles close to the critical angle will result in greaterillumination strength than light propagating at angles far from thecritical angle. To a first approximation, the average illuminationstrength within planar waveguide 800 is inversely proportional tosin(θ_(s,a)), where θ_(s,a) is the incidence angle of propagating lightat the substrate-air interface such that the light is contained withinthe waveguide.

Referring to FIGS. 8 and 9, the manner of coupling light into thewaveguide may be chosen in accordance with the given application. Forexample, the incident light may be coupled into a single layer of themulti-part, planar waveguide, any combination of layers, or all layers.If the light is coupled directly into the low-n interrogation medium,for instance, the light may be inserted into the planar waveguide at anyangle such that Eq. 7 is fulfilled. This range of angles include normalincidence onto the waveguide end (i.e., at an angle perpendicular tosurface normal 820). On the other hand, if the light is coupled inthrough one of the substrates, the angle of incidence should furthersatisfy the conditions:

θ_(s1,m)<(θ_(s1,m))_(c)  [Eq. 8] and

θ_(s2,m)<(θ_(s2,m))_(c).  [Eq. 9]

at the interfaces from first or second substrate 802 and 804 into low-nmedium 810, where the subscript c denotes critical angle. Fulfillment ofthe appropriate one of these conditions ensures that light is eventuallycoupled from the substrate into the low-n medium.

A simple version of the planar low-n index waveguide may be formed fromtwo identical substrates of a single type of material as shown in FIG.9. Alternatively, the two substrates may be non-identical and even becomposed of several disparate layers of optically-clear materials,possibly with different indices of refraction.

Note that, if first or second substrate 802 or 804 is formed of aplurality of disparate layers, the effective refractive index of thecombination of the plurality of disparate layers may be expressed asn_(eff), which is related to the refractive index n_(a) of thesurrounding medium by the equation:

n_(a)<n_(eff).  [Eq. 10]

Furthermore, the two substrates may be in contact with different media,such as if first substrate 802 is exposed to air while second substrate804 is attached to a third substrate (not shown). In this case,multi-part planar waveguide 800 will still work as a waveguide as longas Eqs. 1 and 4 and the additional condition:

n_(a)<n_(m),n_(eff)  [Eq. 11]

are satisfied for both substrates and surrounding media.

The angle of light propagation should be such that the incidence angle θfor the substrate-to-interrogation medium interface, as well as allinterfaces between layers forming the substrate, satisfy the condition:

θ<θ_(c)  [Eq. 12]

and, for interfaces at the substrate and the surrounding medium, theincidence angle θ from the substrate to the surrounding medium shouldfulfill the condition:

θ>θ_(c)  [Eq. 13]

The embodiments illustrated in FIGS. 8-11 impose no constraints on thethicknesses of the interrogation medium or the two substrates as long asthe refractive index and incidence angle requirements of Eqs. 1 and 4are fulfilled. The disclosed embodiments may be particularly suitablefor low-cost, volume production and may be combined with light couplingmechanisms of relatively low complexity. While planar waveguides 800 and900 will function properly with virtually any choice of thicknesses ofthe interrogation medium and substrates, the actual choice of layerthicknesses may be based on a number of factors, such as the choices ofmaterials, manufacturing methods and cost.

The light propagation through thick and thin versions of planarwaveguide 800 is illustrated for both a collimated beam (FIGS. 12 and13) and a diverging beam (FIGS. 14 and 15) as the light input. As shownin FIGS. 12 and 13, a collimated beam 1201 will make distinct passesthrough low-n medium throughout the waveguide with high intensity. For adiverging beam 1401, on the other hand, the reflected light eventuallyoverlaps, resulting in substantially uniform illumination within theplanar waveguide. Consequently, if only one or more,appropriately-placed small regions, extending no more than the portionilluminated by a single pass, require illumination, then collimated beam1201 can provide greater intensity than diverging beam 1401 within thesmall region. If the intent is to illuminate a larger region, possiblyin a uniform fashion, then a diverging beam 1401 may be a better choice.It should also be noted that the pairs of figures (i.e., FIGS. 12-13 andFIGS. 14-15) may be viewed as illustrations of the same planar waveguidebut illuminated with collimated and diverging beams, respectively, ofdifferent beam diameters.

Efficient coupling of light into the waveguide is readily achieved witha combined waveguide thickness of macroscopic extent, e.g., on the orderof few hundreds of nanometers or greater. For instance, a focused laserbeam may be easily coupled into a planar waveguide of such dimensions.The mechanism for appropriately focusing the incoming light may beeither integrated in the waveguide or constructed as a system separatefrom the waveguide. Examples of light coupling mechanisms are shown inFIGS. 16-25.

FIG. 16 shows an embodiment, in which a light beam 1601 is incident atan angle away from surface normal 820 onto second substrate 804. FIG. 17shows a special case, in which a light beam 1701 is directly incident onlow-n medium 810 at an angle perpendicular to surface normal 820. FIG.18 shows a thin, planar waveguide embodiment, in which light beam 1601is simultaneously incident on first and second thin substrates 1802 and1804, respectively, and low-n medium 1810, again at an angle away fromsurface normal 820. FIG. 19 again shows the thin, planar waveguideformed from first and second thin substrates 1802 and 1804,respectively, and low-n medium 1810, with light beam 1701 being insertedinto all three layers at an angle perpendicular to surface normal 820.

FIGS. 20 and 21 show embodiments in which an external lens is used tofocus the incident light beam onto one of the two substrates. FIG. 20shows an embodiment, in which a lens 2010 is used to focus light beam1601 such that a focused beam 2012, which is incident from a non-normalangle away from surface normal 820, is directed into second substrate804. Similarly, FIG. 21 shows an embodiment, in which a light beam 2101,incident at an angle perpendicular to surface normal 820, is focused bya lens 2110 to form a focused beam 2112 before being incident on secondsubstrate 804.

In another approach, the light may be coupled into one of the twosubstrates, which is equipped with an integrated lens assembly forappropriately focusing and directing the incoming light. For instance,FIG. 22 shows an embodiment, in which first and second substrates 2202and 2204, respectively, are spaced apart to contain a low-n medium 2210therebetween. Second substrate 2204 includes an integrated lens 2220,which is configured to receive light beam 1601 so as to couple lightbeam 1601 into second substrate 2204 and, subsequently, the multi-partplanar waveguide configuration. FIG. 23 shows a similar embodiment, inwhich first and second substrates 2302 and 2304, respectively, is spacedapart to contain a low-n medium 2310 therebetween. In this embodiment,second substrate 2304 includes an integrated lens 2320, which is thistime configured to receive light beam 1701, incident at an angleperpendicular to surface normal 820. Light beam 1701, received atintegrated lens 2320, is directed into second substrate 2304 and,subsequently, the multi-part planar waveguide as a whole. FIG. 24 showsan alternative embodiment, which includes first and second substrates2402 and 2404, respectively, separated by first and second gaskets 2406and 2408, respectively, so as to contain a low-n medium 2410therebetween. Second substrate 2404 includes an integrated lens 2420,which is configured to receive light beam 1601 at a portion of secondsubstrate 2404 away from first gasket 2406 such that light beam 1601 isinserted into the multi-part planar waveguide structure without beingblocked by first gasket 2406. Finally, FIG. 25 shows an embodimentincluding first and second substrates 2502 and 2504, respectively. Thistime, rather than including a separate gasket, first substrate 2502includes first and second stand-offs 2506 and 2508, respectively, whichare configured so as to be attachable to second substrate 2504 by, forinstance, laser welding, ultrasonic welding, or other suitable bondingmethod. When bonded together, first and second substrates 2502 and 2504,respectively, defines a volume for containing a low-n medium 2510therebetween. Second substrate 2504 includes an integrated lens 2520configured for receiving light beam 1701, incident at an angleperpendicular to surface normal 820, such that light beam 1701propagates into second substrate 2504 and, subsequently, into themulti-part planar waveguide structure as a whole. Integrated lens 2520may be, for example, an integrated lens as described in theaforementioned U.S. patent application Ser. No. 12/617,535, such thatinsertion of light beam 1701 into second substrate 2504 is substantiallyinsensitive to translation of light beam 1701 with respect to integratedlens 2520.

FIG. 26 shows a side view of an exemplary waveguide structure, shownhere to illustrate insertion, propagation and containment of a lightbeam therethrough. A planar waveguide 2600 includes first and secondsubstrates 2602 and 2604, respectively, spaced apart by first and secondgaskets 2606 and 2608, respectively, so as to contain a low-n medium2610 therein. Second substrate 2604 may optionally include a refractivecomponent, such as an integrated lens 2620 (shown as a dashed curve),for facilitating insertion of a light beam 2630 into planar waveguide2600. As shown in FIG. 26, first and second substrates 2602 and 2604,respectively, low-n medium 2610, and incident angle θ fulfill therefractive index and incident angle conditions specified in Eqs. 1 and 4above such that, after a few TIR bounces at the substrate-airinterfaces, light beam 2630 uniformly illuminates the thickness ofplanar waveguide 2600.

An exemplary embodiment of a cartridge system with interrogation mediumcontainment, in- and outlet ports, and light-coupling means designed forlight entry into the waveguide inside the contained region is shown inFIGS. 27-28. A waveguide cartridge 2700 includes first and secondsubstrates 2702 and 2704, respectively, separated by a gasket 2706 so asto provide containment of a low-n medium 2710 therebetween. Secondsubstrate 2704 includes an integrated lens 2720 for receiving light 2735incident thereon and directing light 2735 into waveguide cartridge 2700so that, after a few TIR bounces therein, light 2735 uniformlyilluminates at least a portion of low-n medium 2710. Waveguide cartridge2700 further includes an inlet port 2742 and an outlet port 2744,through which one or more samples may be introduced into waveguidecartridge 2700 as low-n medium 2710.

The use of optically-clear substrates may facilitate opticalcommunication with the interrogation medium through the substrates. Forinstance, additional image capture through the substrates may beutilized to detect light emitted from the interrogation medium andthereby extracting information about the interrogation medium in, e.g.,microscopy and/or fluorescence applications. Additionally, by using aposition-sensitive detector, spatial information regarding theinterrogation medium may be obtained. Alternatively, light emittedwithin the range of angles confined by the waveguide may be detected inthe plane of the waveguide, if an appropriate pathway is established forallowing this light to exit the waveguide (not shown). For example, amechanism for out-coupling of light may be incorporated into thesubstrate in a manner similar to that used for the in-coupling of light.

As an alternative, one or more of the substrate-surrounding mediuminterfaces may be configured to be at least partially reflective.Additionally, one or more reflecting surfaces may be utilized in thewaveguide. For instance, one or both of the substrate-to-interrogationmedium interfaces may be configured to be partially or completelyreflective in order to better contain the guided light within theinterrogation medium. In the case of configurations wherein the light iscoupled into the waveguide through one of the two substrates, the otherone of the two substrates may be configured to include a reflectivesurface (e.g., at the substrate-to-interrogation medium interface),thereby increasing the illumination intensity within the interrogationmedium. An example of this configuration is shown in FIG. 29, in which awaveguide cartridge 2900 further includes a reflective layer 2910 at theinterface between first substrate 2702 and low-n medium 2710. Theconfiguration as shown in FIG. 29 still allows for optical communicationthrough second substrate 2704 (e.g., for detection of light emitted fromthe interrogation medium), while improving the light containment withinwaveguide cartridge 2900 without affecting the in-coupling of lighttherein. Another advantage of this configuration is a reduced distancefrom light entry to uniform illumination, when guiding a diverging beam.Still another example is shown in FIG. 23, in which a waveguidecartridge 2300 includes a reflective layer 2310 at the interface betweenthe outer surface of first substrate 2702 and surrounding medium 2315.The advantages imparted in the configuration of FIG. 23 is similar tothose discussed in relation to FIG. 29.

Other variations, in which one or both of the substrates include one ormore reflective regions, may hold other advantages. For instance, theconfiguration depicted in FIG. 28 may be modified to include areflective section located at a certain distance from the point of lightentry, thereby reducing the distance required to achieve uniformillumination while maintaining means for optical communication throughboth substrates. Additionally, the at least partially reflectivesurfaces in FIGS. 29 and 30 may be used to direct light emitted by theinterrogation medium (e.g., fluorescence emission) towards a detectorplaced underneath waveguide 100.

While each of the illustrated embodiments shows a single light beamentering the waveguide, the embodiments may be extended to accommodatemultiple beams entering the waveguide. For example, the waveguide may beconstructed to accept multiple beams of light by in-coupling severallight beams through one port, such as a lens integrated into one of thesubstrates, and/or by incorporating several in-coupling ports. The beamsmay propagate in directions that are parallel to each other, either inco- or counter-propagating configurations, or in non-parallelconfigurations.

Example I Detection of Fluorescently Labeled Human Blood Cells

Human peripheral blood mononucleocytes (“PBMCs”) are labeled with CD3Alexa Fluor 647 fluorescence stain, available from InvitrogenCorporation. The cells, whose diameter is 6-12 μm, are kept in a bufferconsisting of phosphate buffered saline with 1% Bovine Serum Albumin and0.06% sodium azide. The buffer with cells is loaded into a cartridge ofthe type shown in FIGS. 27 and 28. The substrate materials and thebuffer lead to a critical angle at the substrate-to-interrogation mediuminterface of θ_(c)=61°. 635 nm laser light is coupled into the systemthrough the curved part of the lower substrate. The curvature isdesigned such that different entry heights result in different angles ofincidence onto the substrate-to-interrogation medium interface. Twodifferent laser heights were used in the present example resulting intwo different angles of incidence onto the substrate-to-interrogationmedium interface: (a) 57° and (b) 66°. With a laser divergence angle of3.5° after passing through the curved surface of the lower substratecase (a) allows the light to pass through the interrogation medium andbe guided by the entire cartridge as shown in FIG. 28. In case (b), onthe other hand, the laser light is confined to the lower substrate andthe interrogation medium is illuminated only by the evanescent field.The 635 nm laser light excites the Alexa Fluor 647 fluorophores and animaging system positioned underneath the cartridge images fluorescenceemitted from the interrogation medium.

TABLE 1 Case (a) Case (b) Using the low-n core Evanescent waveguideconfiguration illumination θ = 57° θ = 66° # cells detected 590 138Staining percentage 56% 13% S/N for representative cell 4.4 1.2

Raw fluorescence images (not shown) indicate that the fluorescence isstrongly enhanced when the interrogation medium is directly illuminated,i.e., case (a). The results are summarized in TABLE 1. In case (a), 590fluorescent cells are detected versus only 138 cells in case (b). Thestaining percentage, i.e., number of fluorescent cells divided by totalnumber of cells, for case (a) agrees with results obtained on a flowcytometer. The signal to noise ratio, S/N, has been calculated as thepeak pixel intensity of a representative cell divided by the standarddeviation of the surrounding background pixel intensities.Alternatively, the signal to noise ratio could have been calculated asthe peak intensity of a cell divided by the background level. However,the former method is the more appropriate parameter when concerned withthe ability to distinguish a cell from the background in the images. Aslisted in TABLE 1, the signal to noise ratio increases almost fourfoldwhen directly illuminating the interrogation medium.

Changes may be made in the above methods and systems without departingfrom the scope hereof. It should thus be noted that the matter containedin the above description or shown in the accompanying drawings should beinterpreted as illustrative and not in a limiting sense. The followingclaims are intended to cover generic and specific features describedherein, as well as statements of the scope of the present method andsystem, which, as a matter of language, might be said to falltherebetween.

Although each of the aforedescribed embodiments have been illustratedwith various components having particular respective orientations, itshould be understood that the system as described in the presentdisclosure may take on a variety of specific configurations with thevarious components being located in a variety of positions and mutualorientations and still remain within the spirit and scope of the presentdisclosure. For example, it should be noted that the presentconfiguration may be applicable for systems in which the core refractiveindex is greater than the refractive indices of the substrates, such asif a solid core material is used, as long as the surrounding mediumrefractive index is less than those of the substrates. Additionally, inthe various figures described above, the gasket may be eliminated andreplaced with direct laser welding of first and second substrates.Furthermore, suitable equivalents may be used in place of or in additionto the various components, the function and use of such substitute oradditional components being held to be familiar to those skilled in theart and are therefore regarded as falling within the scope of thepresent disclosure. Therefore, the present examples are to be consideredas illustrative and not restrictive, and the present disclosure is notto be limited to the details given herein but may be modified within thescope of the appended claims.

What is claimed is:
 1. An apparatus for illuminating a sample, theapparatus comprising: a waveguide including a first substrate includinga first outer surface and a first inner surface, and a second substrateincluding a second outer surface and a second inner surface, the firstand second inner surfaces of the first and second substrates,respectively, being spaced apart from each other and partly defining avolume for confining the sample therein; and a light source forproviding light directed toward the waveguide such that the light isoptically coupled to and contained within the waveguide between theouter surfaces of the first and second substrates, while illuminating atleast a portion of the sample confined within the volume.
 2. Theapparatus of claim 1, the sample containing at least one object, whereinthe waveguide and the light source are configured to cooperate touniformly illuminate the at least one object.
 3. The apparatus of claim2, where in the at least one object is greater than one micrometer indiameter.
 4. The apparatus of claim 1, further comprising a gasket forseparating the first and second inner surfaces of the first and secondsubstrates, respectively, while further defining the volume forconfining the sample therein.
 5. The apparatus of claim 1, wherein thelight is contained between the outer surfaces of the first and secondsubstrates at least in part by total internal reflection.
 6. Theapparatus of claim 1, wherein the light source provides collimatedlight.
 7. The apparatus of claim 6, further comprising a refractiveelement for diverging the collimated light within the planar waveguide.8. The apparatus of claim 1, wherein the first and second surfaces arespaced apart by a distance of more than 10 microns.
 9. The apparatus ofclaim 8, wherein the first and second surfaces are spaced apart by adistance on an order of 100 microns.
 10. The apparatus of claim 1,wherein at least one of the first and second outer surfaces and firstand second inner surfaces is configured for at least partiallyreflecting light incident thereon.
 11. The apparatus of claim 10,wherein the at least one of the first and second outer surfaces andfirst and second inner surfaces is further configured for reflectinglight of a predetermined wavelength range incident thereon.
 12. Theapparatus of claim 1, further comprising a refractive assembly foroptically coupling the light from the light source into the secondsubstrate.
 13. The apparatus of claim 12, wherein the second substrateand the refractive assembly are integrally formed from a single piece ofmaterial.
 14. The apparatus of claim 13, wherein the second substrateand the refractive assembly are formed by injection molding.
 15. Theapparatus of claim 12, wherein the refractive assembly is configuredsuch that a relative translation of the light source in a plane parallelto the inner surface of the second substrate is inconsequential tooptical coupling of the light from the light source to the waveguide.16. The apparatus of claim 12, a surface normal being defined as avector perpendicular to the outer surface of the second substrate,wherein the light is incident on the refractive volume at a non-90°angle away from the surface normal.
 17. A sample analysis systemcomprising: a waveguide including a first substrate including a firstouter surface and a first inner surface, and a second substrateincluding a second outer surface and a second inner surface, the firstand second inner surfaces of the first and second substrates,respectively, being spaced apart from each other and partly defining avolume for confining a sample therein; and a first light source forproviding a first illumination directed toward the waveguide such thatthe first illumination is optically coupled to and contained within thewaveguide between the outer surfaces of the first and second substrateswhile illuminating at least a portion of the sample confined within thevolume; and a detector for detecting a first light signal emitted fromthe sample as a result of the first illumination interacting with theportion of the sample.
 18. The system of claim 17, the sample containingat least one object, wherein the waveguide and the light source areconfigured to cooperate to uniformly illuminate the at least one object.19. The system of claim 18, wherein the at least one object is greaterthan one micrometer in diameter.
 20. The system of claim 17, furthercomprising: a second light source configured for providing a secondillumination; and imaging optics for directing the second illuminationfrom the second light source to at least another portion of the sampleand to the detector, wherein the detector is further configured fordetecting a second light signal resulting from the second illuminationinteracting with the at least another portion of the sample.
 21. Thesystem of claim 17, further comprising a gasket for separating the firstand second inner surfaces of the first and second substrates,respectively, while further defining the volume for confining the sampletherein.
 22. The system of claim 17, wherein the light is containedbetween the outer surfaces of the first and second substrates at leastin part by total internal reflection.
 23. The system of claim 17,wherein the light source provides uncollimated light.
 24. The system ofclaim 17, wherein at least one of the first and second outer surfacesand first and second inner surfaces is configured for at least partiallyreflecting light incident thereon.
 25. The system of claim 24, whereinthe at least one of the first and second outer surfaces and first andsecond inner surfaces is further configured for reflecting light of apredetermined wavelength range incident thereon.
 26. The system of claim17, further comprising a refractive assembly for optically coupling thelight from the light source into the second substrate.
 27. The system ofclaim 26, wherein the second substrate and the refractive assembly areintegrally formed from a single piece of material.
 28. The system ofclaim 27, wherein the second substrate and the refractive assembly areformed by injection molding.
 29. The system of claim 26, wherein therefractive assembly is configured such that a relative translation ofthe light source in a plane parallel to the inner surface of the secondsubstrate is inconsequential to optical coupling of the light from thelight source to the planar waveguide.
 30. The system of claim 26, asurface normal being defined as a vector perpendicular to the outersurface of the second substrate, wherein the light is incident on therefractive volume at an angle away from the surface normal.